Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), infects 1.7 billion people worldwide. Each year, 2-3 million will eventually die of TB. With the discovery of effective anti-tuberculosis drugs, TB became curable. However, Multi Drug Resistant TB and the recent appearance of extensive drug resistant TB, which in many cases is untreatable, has the potential to make TB an incurable disease once again. The TB epidemic has been worsened by HIV/AIDS. Factors involved in the regulation of many phenotypes of MTB such as virulence, latency, antibiotic resistance and ability to survive under harsh conditions are not well understood. Bacterial cell density dependent signaling (quorum sensing), discovered three decades ago, has been shown to play a major role in the control of virulence, sporulation, antibiotic production, resistance to harsh conditions, cell division, biofilm development and drug resistance in bacteria. Data on the role of quorum sensing on the regulation of MTB phenotypes are limited. The isolation and characterization of genes involved in quorum sensing offer a novel approach in understanding and modifying bacterial physiology. In the proposed study, our broad objective is to identify and characterize genes involved in cell-to-cell signaling in mycobacteria. The specific objectives include: (1) To use genetic techniques to study the role of the Rhomboid-like proteins in the physiology of mycobacteria. This will be achieved by inactivation of rhomboid like genes (quorum sensing genes in other organisms) in M. smegmatis and determining how this affects signal production, morphology, survival and other phenotypes of mycobacteria. (2). To construct a library of quorum sensing regulated gene fusions in mycobacteria. This objective will identify the mycobacterial genes whose regulation is cell density dependent. This will be addressed through construction of random lacZ reporter transcriptional gene fusions in M. smegmatis chromosome. (3) To examine the regulatory pathways of the quorum sensing regulated gene fusions in mycobacteria. This will be achieved through screening for other genes that alter the expression of quorum sensing regulated lacZ gene fusions identified in objective 2. This study will provide further information on quorum sensing regulated mycobacterial functions and also describe the regulatory pathways. This will enrich our knowledge on mycobacterial biology and may reveal novel drug and vaccine targets.